Implementing barriers for controlled environments during sample processing and detection

ABSTRACT

Provided herein are methods for processing and/or detecting a sample. A method can comprise providing a barrier between a first region and a second region, wherein the first region comprises the sample, wherein the barrier maintains the first region at a first atmosphere that is different than a second atmosphere of the second region, wherein a portion of the barrier comprises a fluid in coherent motion; and using a detector at least partially contained in the first region to detect one or more signals from the sample while the first region is maintained at the first atmosphere that is different than the second atmosphere of the second region. The portion of the barrier comprising fluid may have a pressure lower than the first atmosphere, the second atmosphere, or both.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/440,026, filed Jun. 13, 2019, which claims the benefit of U.S.Provisional Patent Application No. 62/776,866, filed Dec. 7, 2018, eachof which applications is entirely incorporated herein by reference.

BACKGROUND

Biological sample processing has various applications in the fields ofmolecular biology and medicine (e.g., diagnosis). For example, nucleicacid sequencing may provide information that may be used to diagnose acertain condition in a subject and in some cases tailor a treatmentplan. Sequencing is widely used for molecular biology applications,including vector designs, gene therapy, vaccine design, industrialstrain design and verification. Biological sample processing may involvea fluidics system and/or a detection system.

SUMMARY

Samples, including biologic samples and non-biologic samples, may beprocessed in a controlled environment, such as with a controlledtemperature, pressure, and/or humidity. Analysis of such samples mayinvolve detecting the samples within the controlled environment.Detection may involve continuous detection (e.g., continuous scanning),where there is continuous relative motion between a detector (e.g.,optical head) and a sample. Detection may require proximity between anobjective lens and the sample, such as to achieve direct or indirectcontact between the objective lens and the sample. However, detectionactivities, such as the act of continuously scanning a sample, maydisrupt the controlled environment. In some instances, efforts tomaintain the controlled environment may disrupt the continuous motion ofone or more detectors. In some instances, it may not be possible to movea detector within the controlled environment while maintaining thecontrolled environment because, for example, the presence or motion ofthe detector may make it difficult or impossible to seal or maintain thecontrolled environment, or the presence or motion of the detector mayaffect the sample, thus impacting the detection results. In someinstances, implementing a mechanical seal, such as bellows or slidinggaskets, to maintain the controlled environment from the normalenvironment (e.g., room environment), may introduce unwanted forcesduring the detection and impede or disrupt the relative motion betweenthe detector and the sample. Such problems may yield inaccurate andimprecise detection results. Therefore, recognized herein is a need forsystems, devices, and methods that address at least the abovementionedproblems.

Provided herein are barriers that can be implemented between acontrolled sample environment and the external environment. Suchbarriers may allow for low friction or zero friction relative motionbetween the detector and the sample while maintaining a controlledsample environment. The barriers may allow for an objective lens todirectly or indirectly (e.g., via immersion in a fluid) contact thesample during detection and movement. The barriers may allow forcontinuous scanning involving relative motion in a non-linear direction(e.g., in an R, θ coordinate system) and/or linear direction (e.g., inan X, Y, and/or Z coordinate system). Beneficially, such barriers mayallow for continuous scanning in a 100% or substantially 100% relativehumidity environment. The barriers may prevent humidity from escapingthe sample environment, which when escaped can condense and affect(e.g., corrode, foul, etc.) sensitive equipment, such as the optics andelectronics. Furthermore, the barriers may prevent contaminants from theexternal environment from entering the sample environment, which maycontaminate the sample and/or affect the fluidics and/or detection(e.g., imaging).

A barrier may comprise a transition region between the sampleenvironment and the external environment. The barrier may comprise afluid barrier. The barrier may comprise fluids from the sampleenvironment, the external environment, or both. The barrier may be a lowpressure region. The low pressure region may have lower pressure thanthe sample environment, the external environment, or both. The barriermay comprise a partial vacuum. The barrier may further comprise aphysical barrier.

In an aspect, provided is a method for processing a biological analyte,comprising: (a) providing a barrier between a first region and a secondregion, wherein the first region comprises a substrate having thebiological analyte immobilized adjacent thereto, wherein the barriermaintains the first region at a first atmosphere that is different thana second atmosphere of the second region; and using a detector at leastpartially contained in the first region to detect one or more signals orchanges thereof from the biological analyte while (i) the detector isundergoing translational motion relative to the substrate, wherein thesubstrate and the detector are not in direct mechanical contact, and(ii) the first region is maintained at the first atmosphere that isdifferent than the second atmosphere of the second region.

In some embodiments, a portion of the barrier comprises fluid in bulkmotion. In some embodiments, the portion of the barrier comprises apartial vacuum. In some embodiments, the portion of the barriercomprises fluid from the first region, the second region, or both.

In some embodiments, the first atmosphere is maintained at a firsthumidity that is different than a second humidity of the secondatmosphere. In some embodiments, the first atmosphere has a relativehumidity greater than 90%.

In some embodiments, the detector is an optical detector, and whereinthe one or more signals are one or more optical signals or signalchanges.

In some embodiments, the barrier comprises a first solid component and asecond solid component, wherein the first solid component and the secondsolid component are not in direct mechanical contact, and wherein thefirst solid component is movable relative to the second solid component.

In some embodiments, a portion of the barrier comprises fluid in bulkmotion, and wherein the portion is disposed between the first solidcomponent and the second solid component.

In some embodiments, the detector is fixed relative to the first solidcomponent and wherein the substrate is translationally fixed relative tothe second solid component.

In some embodiments, the substrate is rotatable relative to the secondsolid component.

In some embodiments, a first part of the first solid component isprovided between the first region and the second region, and wherein asecond part of the first solid component is provided between the secondregion and a third region to form part of another barrier configured tomaintain the third region at a third atmosphere that is independent ofthe first atmosphere and the second atmosphere, wherein a portion of theanother barrier comprises fluid in bulk motion, and wherein the thirdregion is movable relative to the first solid component independent ofthe first region.

In some embodiments, the second atmosphere is a room atmosphere or anambient atmosphere.

In some embodiments, a first part of the detector is in the first regionand a second part of the detector is in the second region. In someembodiments, the first part of the detector comprises an optical imagingobjective at least partially immersed in an immersion fluid in contactwith the substrate in the first region.

In some embodiments, the biological analyte is a nucleic acid molecule,and further comprising, based at least in part on the one or moresignals or changes thereof, identifying a sequence of the nucleic acidmolecule or derivative thereof.

In another aspect, provided is a method for processing a biologicalanalyte, comprising: (a) providing a barrier between a first region anda second region, wherein the first region comprises the biologicalanalyte, wherein the barrier maintains the first region at a firstatmosphere that is different than a second atmosphere of the secondregion, wherein a portion of the barrier comprises fluid in bulk motion;and (b) using a detector at least partially contained in the firstregion to detect one or more signals or change thereof from thebiological analyte while the first region is maintained at the firstatmosphere that is different than the second atmosphere of the secondregion.

In some embodiments, the portion of the barrier comprises fluid from thefirst region, the second region, or both.

In some embodiments, the first atmosphere is maintained at a firsthumidity that is different than a second humidity of the secondatmosphere. In some embodiments, the first atmosphere has a relativehumidity greater than 90%.

In some embodiments, (b) comprises moving the detector relative to thebiological analyte while detecting.

In some embodiments, the detector is an optical detector, and whereinthe one or more signals or change thereof are one or more opticalsignals or change thereof.

In some embodiments, the barrier comprises a first solid component and asecond solid component, wherein the first solid component and the secondsolid component are not in mechanical contact, and wherein the firstsolid component is movable relative to the second solid component.

In some embodiments, the portion of the barrier comprising the fluid isdisposed between the first solid component and the second solidcomponent.

In some embodiments, the detector is fixed relative to the first solidcomponent and wherein the biological analyte is translationally fixedrelative to the second solid component.

In some embodiments, a first part of the first solid component isprovided between the first region and the second region, and wherein asecond part of the first solid component is provided between the secondregion and a third region to form part of another barrier configured tomaintain the third region at a third atmosphere that is independent ofthe first atmosphere and the second atmosphere, wherein a portion of theanother barrier comprises fluid, and wherein the third region is movablerelative to the first solid component independent of the first region.

In some embodiments, the second atmosphere is a room atmosphere or anambient atmosphere.

In some embodiments, a first part of the detector is in the first regionand a second part of the detector is in the second region. In someembodiments, the first part of the detector comprises an optical imagingobjective at least partially immersed in an immersion fluid in contactwith the biological analyte in the first region.

In some embodiments, the biological analyte is a nucleic acid molecule,and further comprising, based at least in part on the one or moresignals or signal changes, identifying a sequence of the nucleic acidmolecule or derivative thereof.

In another aspect, provided is a method for processing a biologicalsample, comprising: providing a barrier between a first region and asecond region, wherein the first region comprises the biological sample,wherein the barrier maintains the first region at a first atmospherethat is different than a second atmosphere of the second region, whereina portion of the barrier comprises fluid in coherent motion; and using adetector at least partially contained in the first region to detect oneor more signals from the biological sample while the first region ismaintained at the first atmosphere that is different than the secondatmosphere of the second region.

In some embodiments, the portion of the barrier comprises fluid from thefirst region, the second region, or both.

In some embodiments, the portion of the barrier has a first pressurethat is lower than a second pressure of the first region or a thirdpressure of the second region or both. In some embodiments, the portioncomprises a partial vacuum.

In some embodiments, the first atmosphere is maintained at a firsthumidity that is different than a second humidity of the secondatmosphere. In some embodiments, the first humidity is higher than thesecond humidity. In some embodiments, there is at least a 30% differencebetween the first humidity and the second humidity.

In some embodiments, the first atmosphere has a relative humiditygreater than 90%.

In some embodiments, using said detector comprises moving the detectorrelative to the biological sample while detecting.

In some embodiments, the detector is an optical detector, and whereinthe one or more signals are one or more optical signals.

In some embodiments, the first region comprises a substrate, and whereinthe biological sample is immobilized to the substrate. In someembodiments, the substrate is rotatable.

In some embodiments, the barrier comprises a first solid component and asecond solid component, wherein the first solid component and the secondsolid component are not in mechanical contact, and wherein the firstsolid component is movable relative to the second solid component. Insome embodiments, the portion of the barrier comprising the fluid isdisposed between the first solid component and the second solidcomponent. In some embodiments, the detector is fixed relative to thefirst solid component and wherein the biological sample istranslationally fixed relative to the second solid component.

In some embodiments, a first part of the first solid component isprovided between the first region and the second region, and wherein asecond part of the first solid component is provided between the secondregion and a third region to form part of another barrier configured tomaintain the third region at a third atmosphere that is independent ofthe first atmosphere and the second atmosphere, wherein a portion of theanother barrier comprises fluid, and wherein the third region is movablerelative to the first solid component independent of the first region.

In some embodiments, the second atmosphere is a room atmosphere or anambient atmosphere.

In some embodiments, a first part of the detector is in the first regionand a second part of the detector is in the second region. In someembodiments, the first part of the detector comprises an optical imagingobjective partially immersed in an immersion fluid in contact with thebiological sample in the first region.

In some embodiments, the biological sample is a nucleic acid molecule,and further comprising, based at least in part on the one or moresignals, identifying a sequence of the nucleic acid molecule orderivative thereof.

In another aspect, provided is a method for processing a biologicalsample, comprising: providing a barrier between a first region and asecond region, wherein the first region comprises a substrate having thebiological sample immobilized thereto, wherein the barrier maintains thefirst region at a first atmosphere that is different than a secondatmosphere of the second region; and using a detector at least partiallycontained in the first region to detect one or more signals from thebiological sample while (i) the detector is undergoing translationalmotion relative to the substrate, wherein the substrate and the detectorare not in solid mechanical contact, and (ii) the first region ismaintained at the first atmosphere that is different than the secondatmosphere of the second region.

In some embodiments, a portion of the barrier comprises fluid incoherent motion. In some embodiments, the fluid is from the firstregion, the second region, or both. In some embodiments, the portion ofthe barrier has a first pressure that is lower than a second pressure ofthe first region or a third pressure of the second region or both. Insome embodiments, the portion comprises a partial vacuum.

In some embodiments, the first atmosphere is maintained at a firsthumidity that is different than a second humidity of the secondatmosphere. In some embodiments, the first humidity is higher than thesecond humidity. In some embodiments, there is at least a 30% differencebetween the first humidity and the second humidity.

In some embodiments, the first atmosphere has a relative humiditygreater than 90%.

In some embodiments, the detector is an optical detector, and whereinthe one or more signals are one or more optical signals.

In some embodiments, the barrier comprises a first solid component and asecond solid component, wherein the first solid component and the secondsolid component are not in mechanical contact, and wherein the firstsolid component is movable relative to the second solid component.

In some embodiments, a portion of the barrier comprises fluid incoherent motion, and wherein the portion is disposed between the firstsolid component and the second solid component. In some embodiments, thedetector is fixed relative to the first solid component and wherein thesubstrate is translationally fixed relative to the second solidcomponent. In some embodiments, the substrate is rotatable relative tothe second solid component.

In some embodiments, a first part of the first solid component isprovided between the first region and the second region, and wherein asecond part of the first solid component is provided between the secondregion and a third region to form part of another barrier configured tomaintain the third region at a third atmosphere that is independent ofthe first atmosphere and the second atmosphere, wherein a portion of theanother barrier comprises fluid in coherent motion, and wherein thethird region is movable relative to the first solid componentindependent of the first region.

In some embodiments, the second atmosphere is a room atmosphere or anambient atmosphere.

In some embodiments, a first part of the detector is in the first regionand a second part of the detector is in the second region. In someembodiments, the first part of the detector comprises an optical imagingobjective partially immersed in an immersion fluid in contact with thesubstrate in the first region.

In some embodiments, the biological sample is a nucleic acid molecule,and further comprising, based at least in part on the one or moresignals, identifying a sequence of the nucleic acid molecule orderivative thereof.

In another aspect, provided is a system for processing a biologicalsample, comprising: a barrier disposed between a first region and asecond region, wherein the first region is configured to contain thebiological sample, wherein the barrier is configured to maintain thefirst region at a first atmosphere that is different than a secondatmosphere of the second region, wherein a portion of the barriercomprises a fluid in coherent motion; and a detector at least partiallycontained in the first region, wherein the detector is configured todetect one or more signals from the biological sample while the firstregion is maintained at the first atmosphere that is different than thesecond atmosphere of the second region.

In some embodiments, the portion of the barrier comprises fluid from thefirst region, the second region, or both.

In some embodiments, the portion of the barrier has a first pressurethat is lower than a second pressure of the first region or a thirdpressure of the second region or both. In some embodiments, the portioncomprises a partial vacuum.

In some embodiments, the detector is an optical detector, and whereinthe one or more signals are one or more optical signals.

In some embodiments, the first region comprises a substrate, andwherein, during use, the biological sample is immobilized to thesubstrate.

In some embodiments, the barrier comprises a first solid component and asecond solid component, wherein the first solid component and the secondsolid component are not in mechanical contact, and wherein the firstsolid component is movable relative to the second solid component. Insome embodiments, the portion of the barrier comprising the fluid incoherent motion is disposed between the first solid component and thesecond solid component. In some embodiments, the detector is fixedrelative to the first solid component and wherein, during use, thebiological sample is translationally fixed relative to the second solidcomponent.

In some embodiments, a first part of the first solid component isprovided between the first region and the second region, and wherein asecond part of the first solid component is provided between the secondregion and a third region to form part of another barrier configured tomaintain the third region at a third atmosphere that is independent ofthe first atmosphere and the second atmosphere, wherein a portion of theanother barrier comprises fluid, and wherein the third region is movablerelative to the first solid component independent of the first region.

In some embodiments, the system further comprises a fluid flow unit influid communication with the portion of the barrier, wherein the fluidflow unit is configured to alter or maintain a pressure of the portionof the barrier. In some embodiments, the system further comprises atleast one controller operatively coupled to the detector and the fluidflow unit, wherein the at least one controller is configured to (i)direct the fluid flow unit to alter or maintain the pressure of theportion of the barrier such that the pressure is lower than a firstpressure of the first region, a second pressure of the second region, orboth, and (ii) direct the detector to detect the one or more signals. Insome embodiments, the fluid flow unit comprises one or more fluidchannels through a solid portion of the barrier.

In some embodiments, the system further comprises at least onecontroller operatively coupled to the detector, wherein the at least onecontroller is configured to direct the detector to detect the one ormore signals.

In some embodiments, a first part of the detector is in the first regionand a second part of the detector is in the second region. In someembodiments, the first part of the detector comprises an optical imagingobjective partially immersed in an immersion fluid configured to contactthe biological sample in the first region, during use.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein) of which:

FIG. 1A illustrate a cross-sectional side view of an example barriersystem.

FIG. 1B illustrates a perspective view of FIG. 1A.

FIG. 1C illustrates a cross-sectional view of an example immersionoptical system.

FIG. 2A illustrates a partial cross-sectional view of a barrier systemmaintaining a fluid barrier.

FIG. 2B illustrates a zoomed out view of the barrier system of FIG. 2A.

FIG. 2C illustrates a perspective view of a chamber of the barriersystem of FIG. 2A.

FIG. 3 illustrates a barrier system having multiple sample environments.

FIG. 4 illustrates examples of arrays on a substrate.

FIG. 5 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Provided herein are barriers that can be implemented between acontrolled sample environment and an external environment. A barrier maycomprise a transition region between the sample environment and theexternal environment. The barrier may comprise a fluid barrier. Thebarrier may comprise fluids from the sample environment, the externalenvironment, or both. The barrier may be a low pressure region. The lowpressure region may have lower pressure than the sample environment, theexternal environment, or both. The barrier may comprise a partialvacuum. The barrier may further comprise a physical barrier.

Beneficially, such barriers may allow for zero friction, or lowfriction, relative motion between the detector and the sample whilemaintaining the controlled sample environment. The barriers may allowfor continuous scanning involving relative motion in a non-lineardirection (e.g., in an R, θ coordinate system) and/or linear direction(e.g., in an X, Y, and/or Z coordinate system). The barriers may allowfor continuous scanning in a 100% or substantially 100% relativehumidity environment. The barriers may prevent humidity from escapingthe sample environment, which when escaped can condense and affect(e.g., corrode, foul, etc.) sensitive equipment, such as the optics.Furthermore, the barriers may prevent contaminants from the externalenvironment from entering the sample environment, which may affect thefluidics and/or detection (e.g., imaging).

The term “sample,” as used herein, generally refers to a biologicalsample. The systems, devices, and methods provided herein may beparticularly beneficial for analyzing biological samples, which can behighly sensitive to the environment, such as to the temperature,pressure, and/or humidity of the environment. Biological samples may bederived from any subject or living organism. For example, a subject maybe an animal, a mammal, an avian, a vertebrate, a rodent (e.g., amouse), a primate, a simian, a human, or other organism, such as aplant. Animals may include, but are not limited to, farm animals, sportanimals, and pets. A subject can be a healthy or asymptomaticindividual, an individual that has or is suspected of having a disease(e.g., cancer) or a pre-disposition to the disease, and/or an individualthat is in need of therapy or suspected of needing therapy. A subjectcan be a patient. A subject can be a microorganism or microbe (e.g.,bacteria, fungi, archaea, viruses).

A biological sample may comprise any number of macromolecules, forexample, cellular macromolecules. The biological sample may be a cellsample. The biological sample may be a cell line or cell culture sample.The biological sample can include one or more cells. The biologicalsample can include one or more microbes. The biological sample may be anucleic acid sample or protein sample. The biological sample may also bea carbohydrate sample or a lipid sample. The biological sample may bederived from another sample. The sample may be a tissue sample, such asa biopsy, core biopsy, needle aspirate, or fine needle aspirate. Thesample may be a fluid sample, such as a blood sample, urine sample, orsaliva sample. The sample may be a skin sample. The sample may be acheek swab. The sample may be a plasma or serum sample. The sample maybe a cell-free or cell free sample. A cell-free sample may includeextracellular polynucleotides. Extracellular polynucleotides may beisolated from a bodily sample that may be selected from the groupconsisting of blood, plasma, serum, urine, saliva, mucosal excretions,sputum, stool and tears.

A biological sample may comprise one or more biological particles. Thebiological particle may be a macromolecule. The biological particle maybe a small molecule. The biological particle may be a virus. Thebiological particle may be a cell or derivative of a cell. Thebiological particle may be an organelle. The biological particle may bea rare cell from a population of cells. The biological particle may beany type of cell, including without limitation prokaryotic cells,eukaryotic cells, bacterial, fungal, plant, mammalian, or other animalcell type, mycoplasmas, normal tissue cells, tumor cells, or any othercell type, whether derived from single cell or multicellular organisms.The biological particle may be a constituent (e.g., macromolecularconstituent) of a cell, such as deoxyribonucleic acids (DNA),ribonucleic acids (RNA), nucleus, organelles, proteins, peptides,polypeptides, or any combination thereof. The RNA may be coding ornon-coding. The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) ortransfer RNA (tRNA), for example. The RNA may be a transcript. The RNAmay be small RNA that are less than 200 nucleic acid bases in length, orlarge RNA that are greater than 200 nucleic acid bases in length. SmallRNAs may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA(tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolarRNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA(tsRNA) and small rDNA-derived RNA (srRNA). The RNA may bedouble-stranded RNA or single-stranded RNA. The RNA may be circular RNA.The biological particle may be a hardened cell. Such hardened cell mayor may not include a cell wall or cell membrane. Alternatively or inaddition, samples of the present disclosure may include non-biologicalsamples.

Fluid Barriers

Provided herein are methods for processing and/or detecting a sample. Insome instances, the methods can comprise providing a barrier between afirst region (e.g., sample containing region) and a second region (e.g.,external region), wherein the barrier maintains the first region at afirst atmosphere that is different than a second atmosphere of thesecond region, and wherein a portion of the barrier comprises fluid incoherent motion or bulk motion. The first region can comprise thesample. Then a detector at least partially contained in the first regioncan detect one or more signals from the sample while the first region ismaintained at the first atmosphere that is different than the secondatmosphere of the second region. The detector may not be in directmechanical contact with a substrate contained in the first region,wherein the substrate comprises the sample thereon. The detector may bein fluidic contact with the substrate.

In some instances, the methods can comprise providing a barrier betweena first region (e.g., sample containing region) and a second region(e.g., external region), wherein the barrier maintains the first regionat a first atmosphere that is different than a second atmosphere of thesecond region. The first region can comprise the sample. Then a detectorat least partially contained in the first region can detect one or moresignals from the sample while (i) the detector is undergoing continuouslow friction or zero friction motion relative to the first region, and(ii) the first region is maintained at the first atmosphere that isdifferent than the second atmosphere of the second region. The detectormay not be in direct mechanical contact with a substrate contained inthe first region, wherein the substrate comprises the sample thereon.The detector may be in fluidic contact with the substrate.

Provided herein are systems for processing and/or detecting a sample. Insome instances, the systems can comprise a barrier disposed between afirst region (e.g., sample-containing region) and a second region (e.g.,external region), wherein the first region is configured to contain thesample, wherein the barrier is configured to maintain the first regionat a first atmosphere that is different than a second atmosphere of thesecond region, and wherein a portion of the barrier comprises a fluid incoherent motion or bulk motion. The system can comprise a detector atleast partially contained in the first region, wherein the detector isconfigured to detect one or more signals from the sample while the firstregion is maintained at the first atmosphere that is different than thesecond atmosphere of the second region. In some instances, the detectorcan be configured to detect one or more signals from the sample whilethe detector is undergoing continuous low friction or zero frictionmotion relative to the first region. In some instances, the first regionmay comprise a substrate comprising the sample thereon. For example, thesample may be immobilized adjacent to the substrate. In some instances,the detector may not be in direct mechanical contact with the substrate.In some instances, the detector may be in fluidic contact with thesubstrate.

FIGS. 1A and 1B illustrate an example barrier system 100, showing across-sectional side view and a perspective view, respectively. A fluidbarrier 113 may be implemented between a sample environment 105 (e.g.,first region) and an external environment 107 (e.g., second region). Thesample environment 105 may be a controlled environment, comprising oneor more samples therein. The external environment 107 may be a closed oropen environment. In some instances, the external environment 107 may bea room environment or ambient environment. In some instances, theexternal environment 107 may also be a controlled environment.

The sample environment 105 region may be defined by a chamber 115, aplate 103, and the fluid barrier 113, wherein the fluid barrier 113 ismaintained between a physical gap between the chamber 115 and the plate103. In some instances, the physical gap may be large enough to allowfluid communication between the sample environment 105 and the externalenvironment 107 when the fluid barrier 113 is otherwise not in place.The chamber 115 and the plate 103 may be independent such that thechamber 115, and the sample environment 105 region defined thereby, ismovable relative to the plate 103. For example, the sample environment105 region may be defined by different parts of the plate 103 withdifferent locations of the chamber 115 relative to the plate 103. Therelative motion between the chamber 115 and the plate 103 can be in anydirection, such as in a non-linear direction (e.g., in an R, θcoordinate system) and/or linear direction (e.g., in an X, Y, and/or Zcoordinate system). For example, the relative motion may be rotationalabout a central axis, or linear along any linear axis. In someinstances, actuator units (e.g., linear stages, motors, etc.) and/orstructural units (e.g., beams, supports, tracks, etc.) may constrain therelative motion between the chamber 115 and the plate 103.

The plate 103 and the chamber 115 may not be in direct mechanicalcontact, such that there is a minimal distance between the plate and thechamber. A minimal distance between the plate 103 and the chamber 115may be at least about 100 micrometers (μm), 150 μm, 200 μm, 250 μm, 300μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 millimeter (mm), 2 mm, 3 mm, 4 mm,5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 1 centimeter (cm), or more. Alternativelyor in addition, the minimal distance may be at most about 1 cm, 9 mm, 8mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 950 μm, 900 μm, 850 μm,800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 500 μm, 450 μm, 400 μm,350 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, or less. Alternativelyor in addition, the minimal distance may be within a range defined byany two of the preceding values.

The fluid barrier 113 may act as a transition region between the sampleenvironment 105 and the external environment 107. The fluid barrier 113may comprise fluids (e.g., air) from the sample environment, theexternal environment, or both. The fluid barrier 113 may be a lowpressure region. The fluid barrier 113 may have lower pressure than thesample environment, the external environment, or both. The barrier maycomprise a partial vacuum. In some instances, the fluid barrier 113 maybe a high pressure region. For example, the fluid barrier may have ahigher pressure than the sample environment, the external environment,or both. The fluid barrier 113 may be in coherent motion, such as in acoherent direction of flow. The fluid barrier 113 may be in bulk motion.The fluid barrier may comprise volumes of fluid that has a net averagemotion oriented along one or more directions, or towards a referencedestination. In some instances, a volume of fluid in coherent motion orbulk motion may have stream lines that are oriented along the samegeneral direction. Fluid in coherent motion or bulk motion may bedifferentiated from fluid in random motion that are not part of thefluid barrier (e.g., not in coherent motion, not in bulk motion, nothaving net average motion). Fluid in the fluid barrier may haveturbulent flow and/or laminar flow.

The sample environment 105 may comprise a substrate. One or more samplesmay be immobilized on or adjacent to the substrate. Alternatively or inaddition, the one or more samples may otherwise be disposed on thesubstrate. In some instances, at least a part of the chamber 115 may beor comprise a substrate. In other instances, the chamber 115 may becoupled to a substrate. In some instances, the substrate may be fixedrelative to the chamber 115. Alternatively, the substrate may be movablerelative to the chamber 115, for example, in a linear and/or non-linear(e.g., rotational) direction. For example, the substrate may betranslationally fixed to the chamber 115, but rotatable relative to thechamber 115. Where both the chamber 115 is movable relative to the plate103 and the substrate is movable relative to the chamber 115, the tworelative motions may or may not be operated by the same actuator units.

A detector 101 may protrude into the sample environment 105 from theexternal environment 107 through the plate 103, such as through anaperture in the plate 103. The fit between the detector 101 and theaperture may be fluid-tight such that there is no fluid communicationthrough the aperture when the detector 101 is fitted through theaperture. Alternatively or in addition, the aperture may be hermeticallysealed. Alternatively, the plate 103 may be integral to the detector101. Alternatively, the detector 101 may be entirely contained in thesample environment 105, for example, by affixing a non-sample facing endto the plate 103.

At least a portion of the detector 101 may be fixed relative to theplate 103. In some instances, the detector 101 may be capable oftranslating along an axis that is substantially normal to the plane ofthe plate 103 (e.g., through the aperture) independent of the plate 103.In some instances, at least a portion of the detector 101 (e.g., aportion of the detector inside the sample environment region) may becapable of moving (e.g., linearly or nonlinearly, such as rotating)independent of the plate 103.

Within the sample environment 105, the detector 101 may be configured todetect the one or more samples disposed on the substrate using animmersion optical system, wherein a portion of the detector inside thesample environment 105, such as an optical imaging objective, is inoptical communication with the substrate through a liquid fluid 131medium. In some instances, the liquid fluid medium may be disposed on alocal region of the substrate. In other instances, the liquid fluidmedium may be disposed across an entire area of a surface of thesubstrate (e.g., across a base of chamber 115). Alternatively, thedetector may be in optical communication with the substrate without theliquid fluid medium.

FIG. 1C illustrates a cross-sectional view of an example immersionoptical system 1100. The system 1100 may be used to optically image thesubstrates described herein. The system 1100 may be integrated with anybarrier system described elsewhere herein. The system may comprise anoptical imaging objective 1110 (e.g., detector 101). For example, theobjective may have protruded into the sample environment (e.g., throughplate 103) or may be contained within the sample environment (e.g., andaffixed to a surface of the plate 103). The optical imaging objectivemay be an immersion optical imaging objective. The optical imagingobjective may be configured to be in optical communication with asubstrate 1160. The optical imaging objective may be partially orcompletely surrounded by an enclosure 1120. The enclosure may partiallyor completely surround a sample-facing end of the optical imagingobjective. The enclosure may be fixed to the optical imaging objectiveand/or to the plate. The enclosure may have a generally cup-like shapeor form. The enclosure may be any container. The enclosure may beconfigured to contain a fluid 1140 (such as water or an aqueous solutionor oil or an organic solution) in which the optical imaging objective isto be immersed. The fluid may be in contact with the substrate 1160.

The enclosure 1120 may be configured to maintain a minimal distance 1150between the substrate and the enclosure in order to avoid contactbetween the enclosure and the substrate 1160 during movement of thesubstrate relative to the plate. The minimal distance may be at leastabout 100 nanometers (nm), at 200 nm, 300 nm, 400 nm, 500 nm, 1micrometer (μm), 2 μm, 3 μm, 4 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1 millimeter (mm) or more.Alternatively or in addition, the minimal distance may be at most about1 mm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, 40 μm, 30 μm, 20μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 500 nm, 400 nm, 300 nm, 200 nm,100 nm or less. Alternatively or in addition, the minimal distance maybe within a range defined by any two of the preceding values. Even witha minimal distance, the enclosure may contain the fluid due to surfacetension effects. The system may comprise a fluid flow tube 1130configured to deliver fluid 1140 to the inside of the enclosure. Thefluid flow tube may be connected to the enclosure through an adaptor1135. The adaptor may comprise a threaded adaptor, a compressionadaptor, or any other adaptor.

In some instances, an electrical field application unit (not shown) canbe configured to regulate a hydrophobicity of one or more surfaces of acontainer to retain at least a portion of the fluid contacting theimmersion objective lens and the open substrate, such as by applying anelectrical field.

The optical imaging objective 1110 and enclosure 1120 may provide aphysical barrier between a first location on the substrate in whichchemical processing operations are performed and a second location onthe substrate in which detection operations are performed. In thismanner, the chemical processing operations and the detection operationsmay be performed with independent operation conditions and contaminationof the detector may be avoided. The first and second locations may havedifferent humidities, temperatures, pressures, or atmosphericadmixtures.

FIG. 2A illustrates a partial cross-sectional view of a barrier system200 maintaining a fluid barrier 213. FIG. 2B illustrates a zoomed outview of the barrier system 200. FIG. 2C illustrates a perspective viewof a chamber 215 of the barrier system 200. The barrier system 200,and/or respective components thereof, may correspond to the barriersystem 100, and/or respective components thereof.

The barrier system 200 comprises a sample environment 205 defined by aplate 203, the chamber 215, and the fluid barrier 213. The chamber 215and the plate 203 may be separated by a physical gap. The sampleenvironment 205 may be isolated (and/or insulated) from an externalenvironment 207.

The fluid barrier 213 may act as a transition region between the sampleenvironment 205 and the external environment 207. The fluid barrier 213may comprise fluids (e.g., air) from the sample environment 205, theexternal environment 207, or both. The fluid barrier 213 may be a lowpressure region. The fluid barrier 213 may have lower pressure than thesample environment, the external environment, or both. The fluid barrier213 may be maintained via a fluid flow unit, such as a pressure-alteringapparatus 211. The fluid barrier 213 may comprise fluid in coherentmotion or bulk motion.

The pressure-altering apparatus 211 may be integral to the chamber 215.For example, as illustrated in FIGS. 2A-2C, the pressure-alteringapparatus may be integrated as a fluid channel 220 in a wall of thechamber 215. For example, suction may be applied through the fluidchannel 220 to draw in fluids from the external environment 207, orsample environment 205, or both, to generate a partial vacuum curtain(e.g., in coherent motion, in bulk motion, etc.), thereby creating thefluid barrier 213. The fluid exhaust may be expelled at another end ofthe fluid channel. Alternatively or in addition, the apparatus may notbe integral to the chamber 215. The fluid flow unit and/or thepressure-altering apparatus 211 may be operated via one or morecompressors (e.g., to generate negative pressure), pumps (e.g., togenerate positive pressure), suction apparatus, and/or other devices toprovide the lower pressure in the transition region. The chamber 215 maycomprise one or more fluid channels 220 for implementing fluid barriersof the present disclosure.

While two pressure-altering apparatus 211 is illustrated in FIGS. 2A-2C,it will be appreciated that there may be any number of such apparatus.For example, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50 or more such apparatus. Alternatively or in addition, theremay be at most about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 suchapparatus. In some instances, one or more pressure-altering apparatus211 may be implemented as an annular fluid channel surrounding thesample environment region, or other fluid channel along a perimeter orboundary of the sample environment region.

Beneficially, the fluid barrier 213 may provide a low friction or zerofriction seal between the sample environment 205 and the externalenvironment 207. In some instances, a fluid flow rate through the fluidbarrier 213 may be at least about 5 liters per minute (L/min), 5.5L/min, 6 L/min, 6.5 L/min, 7 L/min, 7.5 L/min, 8 L/min, 8.5 L/min, 9L/min, 9.5 L/min, 10 L/min, 10.5 L/min, 11 L, 11.5 L/min, 12 L/min, 12.5L/min, 13 L/min, 13.5 L/min, 14 L/min, 14.5 L/min, 15 L/min, or more.Alternatively or in addition, the fluid flow rate may be at most about15 L/min, 14.5 L/min, 14 L/min, 13.5 L/min, 13 L/min, 12.5 L/min, 12L/min, 11.5 L/min, 11 L/min, 10.5 L/min, 10 L/min, 9.5 L/min, 9 L/min,8.5 L/min, 8 L/min, 7.5 L/min, 7 L/min, 6.5 L/min, 6 L/min, 5.5 L/min, 5L/min, or less. As will be appreciated the fluid flow rate may vary withdifferent parameters (e.g., minimal distance between the plate andchamber, pressure, temperature, etc.). In an example, for a gap of about500 microns between the plate 203 and the chamber 215, the fluid flowrate can be about 10 L/min or about 13 milliliters per minute (mL/min)per millimeter (mm) along the circumference for a velocity of about 0.42meters per second (m/s).

The systems of the present disclosure may be scaled, such as to havemultiple sample environment regions defined by the same plate. FIG. 3illustrates a barrier system 300 having multiple sample environments.The barrier system 300, and/or respective components thereof, maycorrespond to any other barrier system described herein (e.g., 100and/or 200) and/or respective components thereof.

A single plate 303 may define at least two independent sampleenvironments 305, 309, which are further defined by two independentchambers. Each sample environment may be controlled and maintainedindependent of other sample environments. Each sample environment may bemovable relative to the plate 303 independent of the other sampleenvironments. A fluid barrier may be maintained between each sampleenvironment and the external environment.

While two sample environments are illustrated in FIG. 3, it will beappreciated that systems of the present disclosure may be implementedfor any number of sample environments using a single plate. For example,there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50or more such sample environments in a single plate system. Alternativelyor in addition, there may be at most about 50, 40, 30, 20, 10, 9, 8, 7,6, 5, 4, 3, or 2 such sample environments. Any subset of, or all of, themultiple sample environments may be capable of moving independently ofother sample environments.

In some instances, a single detector in the plate 303 may be used todetect one or more sample environments. Alternatively or in addition, asingle plate 303 may allow at least two detectors to protrude throughthe single plate 303 to detect in parallel. For example, such detectorsmay protrude through the plate via one or more apertures 321 a, 321 bwhich have fluid-tight fits with the detectors. The detectors may befixed relative to the plate. In some instances, the multiple detectorsmay detect two different locations in the same sample environment inparallel. In some instances, the multiple detectors may detect at leasttwo different sample environments in parallel.

While two detector apertures are illustrated in FIG. 3, it will beappreciated that systems of the present disclosure may be implementedfor any number of detectors using a single plate. For example, there maybe at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or moredetectors in a single plate system. Alternatively or in addition, theremay be at most about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 suchdetectors.

The sample environments (e.g., 105, 205, 305, 309) of the presentdisclosure may be controlled. For instance, the environment may bemaintained at a specified temperature or humidity. The environment (orany element thereof) may be maintained at a temperature of at leastabout 20 degrees Celsius (° C.), 25° C., 30° C., 35° C., 40° C., 45° C.,50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C.,95° C., 100° C. or higher. Alternatively, the environment may bemaintained at less than 20° C. Alternatively or in addition, theenvironment (or any element thereof) may be maintained at a temperatureof at most about 100° C., 95° C., 90° C., 85° C., 80° C., 75° C., 70°C., 65° C., 60° C., 55° C., 50° C., 45° C., 40° C., 35° C., 30° C., at25° C., 20° C., or lower. The environment may be maintained at atemperature that is within a range defined by any two of the precedingvalues. Different elements of the sample environment, such as thechamber, protruding portion of the detector, immersion fluid, plate,substrates, solutions, and/or samples therein may be maintained atdifferent temperatures or within different temperature ranges, such asthe temperatures or temperature ranges described herein. Elements of thesystem may be set at temperatures above the dewpoint to preventcondensation. Elements of the system may be set at temperatures belowthe dewpoint to collect condensation.

In some instances, the sample environments may be maintained at higherhumidity than an external environment. In some instances, the sampleenvironments may be maintained at a relative humidity of at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100%. Alternatively or in addition, therelative humidity may be maintained at at most about 100%, 95%, 90%,85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25 20%, 15%,10%, 5%, or less. Alternatively or in addition, the relative humiditymay be maintained within a range defined by any two of the precedingvalues.

An environmental unit (e.g., humidifiers, heaters, heat exchangers,compressors, etc.) may be configured to regulate one or more operatingconditions in each sample environment. In some instances, eachenvironment may be regulated by independent environmental units. In someinstances, a single environmental unit may regulate a plurality ofenvironments. In some instances, a plurality of environmental units may,individually or collectively, regulate the different environments. Anenvironmental unit may use active methods or passive methods to regulatethe operating conditions. For example, the temperature may be controlledusing heating or cooling elements. The humidity may be controlled usinghumidifiers or dehumidifiers. In some instances, a first part of thesample environment may be further controlled from other parts of thesample environment. Different parts may have different localtemperatures, pressures, and/or humidity. For example, the sampleenvironment may comprise a first internal environment and a secondinternal environment separated by a seal. In some instances, the sealmay comprise an immersion objective lens, as described elsewhere herein.For example, the immersion objective lens may be part of a seal thatseparates the sample environment into a first internal environmenthaving 100% (or substantially 100%) relative humidity and a secondenvironment having a different temperature, pressure or humidity. Thesecond environment may or may not be an ambient environment. Theimmersion objective lens may be in contact a detector.

External environments (e.g., 107, 207) of the present disclosure may beany environment external to the sample environments. For example, theexternal environment may be a room environment. The external environmentmay be an ambient environment. The external environment may itself becontrolled, such as via one or more environmental units describedelsewhere herein. The external environment may be open or closed. Insome instances, the external environment may be at room temperature,pressure, and/or humidity. In some instances, the external environmentmay be at ambient temperature, pressure, and/or humidity.

Chambers (e.g., 115, 215) of the present disclosure may comprise a baseand side walls to define an opening that nearly contacts the plate. Theside walls may be a closed continuous surface, or a plurality ofadjacent (and/or adjoining) surfaces. For example, the base may compriseor be the substrate. In some instances, the base may be coupled to thesubstrate. The substrate may be translationally fixed to the base, butrotatable relative to the base. In some instances, at least a portion ofa side wall of the chamber may have thickness dimensions large enough toallow integration of one or more fluid channels to allow operation ofthe pressure-altering apparatus. In some instances, a side wall of thechamber may have thickness dimensions large enough to maintain the lowpressure fluid barrier. The chamber may entirely or partially compriseone or more of glass, silicon, a metal such as aluminum, copper,titanium, chromium, or steel, a ceramic such as titanium oxide orsilicon nitride, a plastic such as polyethylene (PE), low-densitypolyethylene (LDPE), high-density polyethylene (HDPE), polypropylene(PP), polystyrene (PS), high impact polystyrene (HIPS), polyvinylchloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile butadienestyrene (ABS), polyacetylene, polyamides, polycarbonates, polyesters,polyurethanes, polyepoxide, polymethyl methacrylate (PMMA),polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamineformaldehyde (MF), urea-formaldehyde (UF), polyetheretherketone (PEEK),polyetherimide (PEI), polyimides, polylactic acid (PLA), furans,silicones, polysulfones, any mixture of any of the preceding materials,or any other appropriate material.

Substrates of the present disclosure may be an open substrate. Thesubstrate may be a solid substrate. The substrate may entirely orpartially comprise one or more of glass, silicon, a metal such asaluminum, copper, titanium, chromium, or steel, a ceramic such astitanium oxide or silicon nitride, a plastic such as polyethylene (PE),low-density polyethylene (LDPE), high-density polyethylene (HDPE),polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS),polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrilebutadiene styrene (ABS), polyacetylene, polyamides, polycarbonates,polyesters, polyurethanes, polyepoxide, polymethyl methacrylate (PMMA),polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamineformaldehyde (MF), urea-formaldehyde (UF), polyetheretherketone (PEEK),polyetherimide (PEI), polyimides, polylactic acid (PLA), furans,silicones, polysulfones, any mixture of any of the preceding materials,or any other appropriate material. The substrate may be entirely orpartially coated with one or more layers of a metal such as aluminum,copper, silver, or gold, an oxide such as a silicon oxide (Si_(x)O_(y),where x, y may take on any possible values), a photoresist such as SU8,a surface coating such as an aminosilane or hydrogel, polyacrylic acid,polyacrylamide dextran, polyethylene glycol (PEG), or any combination ofany of the preceding materials, or any other appropriate coating. Theone or more layers may have a thickness of at least 1 nanometer (nm), atleast 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500μm, or at least 1 millimeter (mm). The one or more layers may have athickness that is within a range defined by any two of the precedingvalues.

The substrate and/or chamber may have any shape, form or dimension. Insome instances, for example, the substrate may have the general form ofa cylinder, a cylindrical shell or disk, a rectangular prism, or anyother geometric form. The substrate may have a thickness (e.g., aminimum dimension) of at least about 100 μm, 200 μm, 300 μm, 400 μm, 500μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm or more.The substrate may have a thickness that is within a range defined by anytwo of the preceding values. The substrate may have a first lateraldimension (such as a width for a substrate having the general form of arectangular prism or a radius for a substrate having the general form ofa cylinder) of at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 1 cm, 2 cm,3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 20 cm, 30 cm, 40 cm, 50cm, 60 cm, 70 cm, 80 cm, 90 cm, 1 meter (m) or more. The substrate mayhave a first lateral dimension that is within a range defined by any twoof the preceding values. The substrate may have a second lateraldimension (such as a length for a substrate having the general form of arectangular prism) or at least at least about 1 mm, 2 mm, 3 mm, 4 mm, 5mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 20 cm,30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, 1 meter (m) or more.The substrate may have a second lateral dimension that is within a rangedefined by any two of the preceding values. A surface of the substratemay be planar or substantially planar. Alternatively or in addition to,a surface of the substrate may be textured or patterned. For example,the substrate may comprise grooves, troughs, hills, and/or pillars. Insome instances, the substrate may comprise wells. In some instances, thesubstrate may define one or more cavities (e.g., micro-scale cavities ornano-scale cavities). The substrate may have a regular textures and/orpatterns across the surface of the substrate. For example, the substratemay have regular geometric structures (e.g., wedges, cuboids, cylinders,spheroids, hemispheres, etc.) above or below a reference level of thesurface. Alternatively, the substrate may have irregular textures and/orpatterns across the surface of the substrate. For example, the substratemay have any arbitrary structure above or below a reference level of thesubstrate. In some instances, a texture of the substrate may comprisestructures having a maximum dimension of at most about 100%, 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% of the total thickness of thesubstrate or a layer of the substrate. In some instances, the texturesand/or patterns of the substrate may define at least part of anindividually addressable location on the substrate. A textured and/orpatterned substrate may be substantially planar.

The substrate may comprise an array. For instance, the array may belocated on a lateral surface of the substrate. The array may be a planararray. The array may have the general shape of a circle, annulus,rectangle, or any other shape. The array may comprise linear and/ornon-linear rows. The array may be evenly spaced or distributed. Thearray may be arbitrarily spaced or distributed. The array may haveregular spacing. The array may have irregular spacing. The array may bea textured array. The array may be a patterned array. FIG. 4 illustratesexamples of arrays of individually addressable locations 401 on asubstrate (e.g., from a top view), with panel A showing a substantiallyrectangular substrate with regular linear arrays, panel B showing asubstantially circular substrate with regular linear arrays, and panel Cshowing an arbitrarily shaped substrate with irregular arrays.

The array may comprise a plurality of individually addressable locations(e.g., 401). In some instances, the locations may correspond toindividually addressable coordinates on the substrate. Alternatively orin addition, the locations may correspond to physical structures (e.g.,wells) on the substrate. An analyte to be processed and/or detected bythe detector may be immobilized to the array. The array may comprise oneor more binders described herein, such as one or more physical linkersor adapters or chemical linkers or adapters that are coupled to, orconfigured to couple to, an analyte. For instance, the array maycomprise a linker or adaptor that is coupled to a nucleic acid molecule.Alternatively or in addition to, the analyte may be coupled to a bead,and the bead may be immobilized to the array.

The individually addressable locations may comprise locations ofanalytes or groups of analytes that are accessible for manipulation. Themanipulation may comprise placement, extraction, reagent dispensing,seeding, heating, cooling, or agitation. The extraction may compriseextracting individual analytes or groups of analytes. For instance, theextraction may comprise extracting at least 2, at least 5, at least 10,at least 20, at least 50, at least 100, at least 200, at least 500, orat least 1,000 analytes or groups of analytes. Alternatively or inaddition to, the extraction may comprise extracting at most 1,000, atmost 500, at most 200, at most 100, at most 50, at most 20, at most 10,at most 5, or at most 2 analytes or groups of analytes. The manipulationmay be accomplished through, for example, localized microfluidic, pipet,optical, laser, acoustic, magnetic, and/or electromagnetic interactionswith the analyte or its surroundings.

The array may be coated with binders. For instance, the array may berandomly coated with binders. Alternatively, the array may be coatedwith binders arranged in a regular pattern (e.g., in linear arrays,radial arrays, hexagonal arrays etc.). The array may be coated withbinders on at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% of the number ofindividually addressable locations, or of the surface area of thesubstrate. The array may be coated with binders on a fraction ofindividually addressable locations, or of the surface areas of thesubstrate, that is within a range defined by any two of the precedingvalues. The binders may be integral to the array. The binders may beadded to the array. For instance, the binders may be added to the arrayas one or more coating layers on the array.

The binders may immobilize analytes through non-specific interactions,such as one or more of hydrophilic interactions, hydrophobicinteractions, electrostatic interactions, physical interactions (forinstance, adhesion to pillars or settling within wells), and the like.In some instances, the binders may immobilize biological analytesthrough specific interactions. For instance, where a biological analyteis a nucleic acid molecule, the binders may comprise oligonucleotideadaptors configured to bind to the nucleic acid molecule. Alternativelyor in addition, such as to bind other types of analytes, the binders maycomprise one or more of antibodies, oligonucleotides, aptamers, affinitybinding proteins, lipids, carbohydrates, and the like. The binders mayimmobilize biological analytes through any possible combination ofinteractions. For instance, the binders may immobilize nucleic acidmolecules through a combination of physical and chemical interactions,through a combination of protein and nucleic acid interactions, etc. Thearray may comprise at least about 10, 100, 1000, 10,000, 100,000,1,000,000, 10,000,000, 100,000,000 or more binders. Alternatively or inaddition, the array may comprise at most about 100,000,000, 10,000,000,1,000,000, 100,000, 10,000, 1000, 100, 10 or fewer binders. The arraymay have a number of binders that is within a range defined by any twoof the preceding values. In some instances, a single binder may bind asingle analyte (e.g., nucleic acid molecule). In some instances, asingle binder may bind a plurality of analytes (e.g., plurality ofnucleic acid molecules). In some instances, a plurality of binders maybind a single analyte. Though some examples herein describe interactionsof binders with nucleic acid molecules, the binders may immobilize othermolecules (such as proteins), other particles, cells, viruses, otherorganisms, or the like, and non-biological analytes.

In some instances, each location, or a subset of such locations, mayhave immobilized thereto an analyte (e.g., a nucleic acid molecule, aprotein molecule, a carbohydrate molecule, etc.). In other instances, afraction of the plurality of individually addressable location may haveimmobilized thereto an analyte. A plurality of analytes immobilized tothe substrate may be copies of a template analyte. For example, theplurality of analytes (e.g., nucleic acid molecules) may have sequencehomology. In other instances, the plurality of analytes immobilized tothe substrate may not be copies. The plurality of analytes may be of thesame type of analyte (e.g., a nucleic acid molecule) or may be acombination of different types of analytes (e.g., nucleic acidmolecules, protein molecules, etc.).

In some instances, the array may comprise a plurality of types ofbinders, such as to bind different types of analytes. For example, thearray may comprise a first type of binders (e.g., oligonucleotides)configured to bind a first type of analyte (e.g., nucleic acidmolecules), and a second type of binders (e.g., antibodies) configuredto bind a second type of analyte (e.g., proteins), and the like. Inanother example, the array may comprise a first type of binders (e.g.,first type of oligonucleotide molecules) to bind a first type of nucleicacid molecules and a second type of binders (e.g., second type ofoligonucleotide molecules) to bind a second type of nucleic acidmolecules, and the like. For example, the substrate may be configured tobind different types of analytes in certain fractions or specificlocations on the substrate by having the different types of binders inthe certain fractions or specific locations on the substrate.

An analyte may be immobilized to the array at a given individuallyaddressable location of the plurality of individually addressablelocations. An array may have any number of individually addressablelocations. For instance, the array may have at least 1, at least 2, atleast 5, at least 10, at least 20, at least 50, at least 100, at least200, at least 500, at least 1,000, at least 2,000, at least 5,000, atleast 10,000, at least 20,000, at least 50,000, at least 100,000, atleast 200,000, at least 500,000, at least 1,000,000, at least 2,000,000,at least 5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, at least 1,000,000,000, at least 2,000,000,000, at least5,000,000,000, at least 10,000,000,000, at least 20,000,000,000, atleast 50,000,000,000, or at least 100,000,000,000 individuallyaddressable locations. The array may have a number of individuallyaddressable locations that is within a range defined by any two of thepreceding values. Each individually addressable location may bedigitally and/or physically accessible individually (from the pluralityof individually addressable locations). For example, each individuallyaddressable location may be located, identified, and/or accessedelectronically or digitally for mapping, sensing, associating with adevice (e.g., detector, processor, dispenser, etc.), or otherwiseprocessing. Alternatively or in addition to, each individuallyaddressable location may be located, identified, and/or accessedphysically, such as for physical manipulation or extraction of ananalyte, reagent, particle, or other component located at anindividually addressable location.

Each individually addressable location may have the general shape orform of a circle, rectangle, pit, bump, or any other shape or form. Eachindividually addressable location may have a first lateral dimension(such as a radius for individually addressable locations having thegeneral shape of a circle or a width for individually addressablelocations having the general shape of a rectangle). The first lateraldimension may be at least 1 nanometer (nm), at least 2 nm, at least 5nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, atleast 200 nm, at least 500 nm, at least 1,000 nm, at least 2,000 nm, atleast 5,000 nm, or at least 10,000 nm. The first lateral dimension maybe within a range defined by any two of the preceding values. Eachindividually addressable location may have a second lateral dimension(such as a length for individually addressable locations having thegeneral shape of a rectangle). The second lateral dimension may be atleast 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, atleast 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least500 nm, at least 1,000 nm, at least 2,000 nm, at least 5,000 nm, or atleast 10,000 nm. The second lateral dimension may be within a rangedefined by any two of the preceding values. In some instances, eachindividually addressable locations may have or be coupled to a binder,as described herein, to immobilize a analyte thereto. In some instances,only a fraction of the individually addressable locations may have or becoupled to a binder. In some instances, an individually addressablelocation may have or be coupled to a plurality of binders to immobilizean analyte thereto.

The analytes bound to the individually addressable locations mayinclude, but are not limited to, molecules, cells, organisms, nucleicacid molecules, nucleic acid colonies, beads, clusters, polonies, or DNAnanoballs. The bound analytes may be immobilized to the array in aregular, patterned, periodic, random, or pseudo-random configuration, orany other spatial arrangement.

While examples of the present disclosure describe the processing and/ordetection of samples and analytes immobilized to individuallyaddressable locations on a substrate, the systems, devices, and methodsdescribed herein also allows for detection of the substrate itself(without any samples and/or analytes disposed thereon).

The substrate may be configured to move with respect to the plate. Suchmotion may be facilitated by one or more actuators or other devices(e.g., gears, stages, actuators, discs, pulleys, motors, etc.). Suchactuators and devices may be mechanically connected to the substratedirectly or indirectly via intermediary components. Such actuators anddevices may be automated. Alternatively or in addition, the actuatorsand devices may receive manual input. The substrate may be configured tomove at any speed that allows for detection. In some instances, orrotational motion, the axis of rotation may be an axis through thecenter of the substrate. The axis may be an off-center axis. Forinstance, the substrate may be affixed to a chuck (such as a vacuumchuck). The substrate may be configured to rotate with a rotationalvelocity of at least 1 revolution per minute (rpm), at least 2 rpm, atleast 5 rpm, at least 10 rpm, at least 20 rpm, at least 50 rpm, at least100 rpm, at least 200 rpm, at least 500 rpm, at least 1,000 rpm, atleast 2,000 rpm, at least 5,000 rpm, or at least 10,000 rpm. Thesubstrate may be configured to rotate with a rotational velocity that iswithin a range defined by any two of the preceding values. The substratemay be configured to rotate with different rotational velocities duringdifferent operations described herein. The substrate may be configuredto rotate with a rotational velocity that varies according to atime-dependent function, such as a ramp, sinusoid, pulse, or otherfunction or combination of functions. The time-varying function may beperiodic or aperiodic.

The fluid barriers provided herein may provide zero friction or lowfriction relative motion between the substrate and the detector. Theremay be no mechanical contact between the plate (coupled to the detector)and the chamber (coupled to the substrate).

Detectors (e.g., 101, 1110) of the present disclosure may includedevices that are capable of detecting a signal. For example, the signalcan be a signal indicative of the presence or absence of one or morecomponents (e.g., incorporated nucleotides, fluorescent labels,electronic signals, etc.) and/or a signal indicative of a change ofstate in one or more components. The detector may detect multiplesignals. The signal or multiple signals may be detected in real-time,prior to, during (or substantially during), or subsequent to a reaction,such as a sequencing reaction. In some cases, a detector can includeoptical and/or electronic components that can detect signals. A detectormay implement one or more detection methods. Non-limiting examples ofdetection methods include optical detection, spectroscopic detection,electrostatic detection, electrochemical detection, acoustic detection,magnetic detection, and the like. Optical detection methods include, butare not limited to, light absorption, ultraviolet-visible (UV-vis) lightabsorption, infrared light absorption, light scattering, Rayleighscattering, Raman scattering, surface-enhanced Raman scattering, Miescattering, fluorescence, luminescence, and phosphorescence.Spectroscopic detection methods include, but are not limited to, massspectrometry, nuclear magnetic resonance (NMR) spectroscopy, andinfrared spectroscopy. Electrostatic detection methods include, but arenot limited to, gel based techniques, such as, for example, gelelectrophoresis. Electrochemical detection methods include, but are notlimited to, electrochemical detection of amplified product afterhigh-performance liquid chromatography separation of the amplifiedproducts.

A detectable signal, such as an optical signal (e.g., fluorescentsignal), may be generated upon reaction an analyte and another component(e.g., a probe). For example, the signal may originate from the probeand/or the analyte. The detectable signal may be indicative of areaction or interaction between the probe and the analyte. Thedetectable signal may be a non-optical signal. For example, thedetectable signal may be an electronic signal. The detectable signal maybe detected by one or more sensors. For example, an optical signal maybe detected via one or more optical detectors in an optical detectionscheme described elsewhere herein. The signal may be detected duringmotion of the substrate. The signal may be detected followingtermination of the motion. In some instances, after the detection, thesignal may be muted, such as by cleaving a label from the probe and/orthe analyte, and/or modifying the probe and/or the analyte. Suchcleaving and/or modification may be effected by one or more stimuli,such as exposure to a chemical, an enzyme, light (e.g., ultravioletlight), or temperature change (e.g., heat). In some instances, thesignal may otherwise become undetectable by deactivating or changing themode (e.g., detection wavelength) of the one or more sensors, orterminating or reversing an excitation of the signal. In some instances,detection of a signal may comprise capturing an image or generating adigital output (e.g., between different images).

The detectors may be capable of continuous area scanning, duringcontinuous linear motion and/or a continuous non-linear (e.g.,rotational) motion between the sample and the substrate. For example,the detectors can scan a substrate or array along a linear orsubstantially linear path. Alternatively or in addition, the detectorsmay scan along a nonlinear path, including in rings, spirals, or arcs ona rotating substrate. The detector may be a continuous area scanningdetector. A continuous area scanning detector may comprise an imagingarray sensor capable of continuous integration over a scanning areawherein the scanning is electronically synchronized to the image of anobject in relative motion. A continuous area scanning detector maycomprise a time delay and integration (TDI) charge coupled device (CCD),Hybrid TDI, and/or complementary metal oxide semiconductor (CMOS) pseudoTDI.

For rotational scan paths, the scanning direction may be substantially θin an (R, θ) coordinate system in which the object rotation motion is ina θ direction. Across any field of view on the object (substrate) imagedby a scanning system, the apparent velocity may vary with the radialposition (R) of the field point on the object as R dθ/dt. Continuousarea scanning detectors may scan at the same rate for all imagepositions and therefore may not be able to operate at the correct scanrate for all imaged points in a curved (or arcuate or non-linear) scan.Therefore the scan may be corrupted by velocity blur for imaged fieldpoints moving at a velocity different than the scan velocity. Continuousrotational area scanning may comprise an optical detection system ormethod that makes algorithmic, optical, and/or electronic corrections tosubstantially compensate for this tangential velocity blur, therebyreducing this scanning aberration. For example, the compensation isaccomplished algorithmically by using an image processing algorithm thatdeconvolves differential velocity blur at various image positionscorresponding to different radii on the rotating substrate to compensatefor differential velocity blur. In another example, the compensation isaccomplished by using an anamorphic magnification gradient. This mayserve to magnify the substrate in one axis (anamorphic magnification) bydifferent amounts at two or more substrate positions transverse to thescan direction. The anamorphic magnification gradient may modify theimaged velocities of the two or more positions to be substantially equalthereby compensating for tangential velocity differences of the twopositions on the substrate. This compensation may be adjustable toaccount for different velocity gradients across the field of view atdifferent radii on the substrate. In some instances, the imaging fieldof view may be segmented into two or more regions, each of which can beelectronically controlled to scan at a different rate. These rates maybe adjusted to the mean projected object velocity within each region.The regions may be optically defined using one or more beam splitters orone or more mirrors. The two or more regions may be directed to two ormore detectors. The regions may be defined as segments of a singledetector.

The systems, devices, and methods described herein may have particularbiological applications. In an example, the fluid barrier systems may beused in nucleic acid sequencing applications. A sample environment maybe provided within a chamber having a substrate comprising an array. Aplurality of nucleic acid molecules may be immobilized to individuallyaddressable locations in the array. A solution of labeled nucleotidesmay be dispensed to the substrate under conditions sufficient to allowincorporation of at least a subset of the labeled nucleotides into atleast a subset of the plurality of nucleic acid molecules, ifappropriate (e.g., labeled nucleotides are complementary to an openposition in the nucleic acid molecules), and the unincorporatednucleotides washed with a washing solution. The sample environment,including temperature, pressure, and/or humidity, may be maintained inaccordance with the particular samples (e.g., nucleic acid molecules)used and/or processing (e.g., incorporation reactions) carried out inthe sample environment. Then, while implementing the fluid barriers andthereby maintaining the sample environment conditions, a detectorprotruding through a plate into the sample environment, configured asdescribed elsewhere herein, may detect one or more detectable signalsfrom the incorporated labeled nucleotides from the individuallyaddressable locations in the array during relative motion of thedetector and the substrate. For example, the substrate may be movedrelative to the detector such as to allow the detector detects allindividually addressable locations in (or a desired sub-area of) thesubstrate. In some instances, the substrate may undergo a rotationalmotion and a then a linear motion, in repeated cycles, such that aftereach rotational motion, the detector is able to scan an annular ring,and after each linear motion, the detector is positioned to scan anotherannular ring at a different radius from a center of the substrate.Alternatively or in addition, the substrate may undergo only rotationalmotion. Alternatively or in addition, the substrate may undergo onlylinear motion.

The fluid barriers maintained during the detection may provide barriersbetween the controlled sample environment and the external environment,and allow for low friction or zero friction relative motion between thedetector and the sample, while maintaining a controlled sampleenvironment. Beneficially, such barriers may allow for continuousscanning in a 100% or substantially 100% relative humidity environment.The barriers may prevent humidity from escaping the sample environment,which when escaped can condense and affect (e.g., corrode, foul, etc.)sensitive equipment, such as the optics. Furthermore, the barriers mayprevent contaminants from the external environment from entering thesample environment, which may affect the fluidics and/or detection(e.g., imaging).

As will be appreciated, the systems, devices, and methods describedherein may also have non-biological applications, such as for analyzingnon-biological samples.

Computer Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 5 shows acomputer system 501 that is programmed or otherwise configured toprocess and/or detect a sample. The computer system 501 can regulatevarious aspects of methods and systems of the present disclosure. Forexample, the computer system 501 may comprise, or be, a controllerconfigured to communicate with the fluid flow unit, actuators, and/ordetectors of the systems described herein.

The computer system 501 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 505, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 501 also includes memory or memorylocation 510 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 515 (e.g., hard disk), communicationinterface 520 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 525, such as cache, other memory,data storage and/or electronic display adapters. The memory 510, storageunit 515, interface 520 and peripheral devices 525 are in communicationwith the CPU 505 through a communication bus (solid lines), such as amotherboard. The storage unit 515 can be a data storage unit (or datarepository) for storing data. The computer system 501 can be operativelycoupled to a computer network (“network”) 530 with the aid of thecommunication interface 520. The network 530 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 530 in some cases is atelecommunication and/or data network. The network 530 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. The network 530, in some cases with the aid of thecomputer system 501, can implement a peer-to-peer network, which mayenable devices coupled to the computer system 501 to behave as a clientor a server.

The CPU 505 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 510. The instructionscan be directed to the CPU 505, which can subsequently program orotherwise configure the CPU 505 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 505 can includefetch, decode, execute, and writeback.

The CPU 505 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 501 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 515 can store files, such as drivers, libraries andsaved programs. The storage unit 515 can store user data, e.g., userpreferences and user programs. The computer system 501 in some cases caninclude one or more additional data storage units that are external tothe computer system 501, such as located on a remote server that is incommunication with the computer system 501 through an intranet or theInternet.

The computer system 501 can communicate with one or more remote computersystems through the network 530. For instance, the computer system 501can communicate with a remote computer system of a user. Examples ofremote computer systems include personal computers (e.g., portable PC),slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab),telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device,Blackberry®), or personal digital assistants. The user can access thecomputer system 501 via the network 530.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 501, such as, for example, on the memory510 or electronic storage unit 515. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 505. In some cases, the code canbe retrieved from the storage unit 515 and stored on the memory 510 forready access by the processor 505. In some situations, the electronicstorage unit 515 can be precluded, and machine-executable instructionsare stored on memory 510.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 501, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 501 can include or be in communication with anelectronic display 535 that comprises a user interface (UI) 540 forproviding, for example, detection results to a user. The UI may furtherpresent a console for configuring the fluid barrier systems, and/orcomponents thereof (e.g., pressure-altering apparatus, environmentalunits, detectors, immersion enclosure, motion of detectors, motion ofplates, motion of containers, motion of substrates, sample processing,etc.) of the present disclosure. Examples of UI's include, withoutlimitation, a graphical user interface (GUI) and web-based userinterface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 505.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A system for processing or analyzing an analyte, comprising: a chamber and a lid, wherein said chamber comprises a first region configured to contain (1) a substrate comprising said analyte immobilized adjacent thereto, and (2) at least a portion of a detection unit, and wherein said lid is configured to be disposed adjacent to said chamber; and a fluid flow unit configured to provide fluid in bulk motion at a location disposed between said chamber and said lid when said lid is disposed adjacent to said chamber, such that said first region is maintained at a first atmosphere that is different than a second atmosphere of a second region external to said first region.
 2. The system of claim 1, wherein said fluid in bulk motion is configured to provide a partial vacuum between said chamber and said lid.
 3. The system of claim 1, wherein said fluid flow unit is configured to use fluid from said first region, said second region, or both to provide said fluid in bulk motion.
 4. The system of claim 1, wherein said fluid comprises air.
 5. The system of claim 1, wherein said fluid flow unit is configured to maintain said first region at a first humidity or first humidity range, wherein said first humidity or first humidity range is different than a second humidity or second humidity range of said second region.
 6. The system of claim 5, wherein said first atmosphere has a relative humidity greater than 90%.
 7. The system of claim 1, wherein said fluid flow unit is configured to maintain said first region at a first temperature or first temperature range, wherein said first temperature or first temperature range is different than a second temperature or second temperature range of said second region.
 8. The system of claim 1, wherein said first region comprises a first part and a second part, wherein said fluid flow unit is configured to maintain said first part at a first local atmosphere and maintain said second part at a second local atmosphere different than said first local atmosphere.
 9. The system of claim 8, wherein said fluid flow unit is configured to maintain said first local atmosphere at a first local temperature or first local temperature range that is different than a second local temperature or second local temperature range of said second local atmosphere.
 10. The system of claim 8, wherein said fluid flow unit is configured to maintain said first local atmosphere at a first local humidity or first local humidity range that is different than a second local humidity or second local humidity range of said second local atmosphere.
 11. The system of claim 1, wherein said detection unit is at least partially contained in said first region.
 12. The system of claim 11, wherein said detection unit is an optical detection unit.
 13. The system of claim 11, wherein a first portion of said detection unit is in said first region and a second portion of said detection unit is in said second region.
 14. The system of claim 11, wherein said first portion of said detection unit comprises an optical imaging objective that is configured to be at least partially immersed in an immersion fluid in contact with said substrate in said first region.
 15. The system of claim 11, wherein said detection unit is configured to undergo motion while said substrate is stationary.
 16. The system of claim 11, wherein said substrate is configured to undergo motion while said detection unit is stationary.
 17. The system of claim 11, wherein said relative motion comprises one or more members selected from the group consisting of (i) substantially linear motion and (ii) substantially non-linear motion.
 18. The system of claim 11, wherein said detection unit is configured to be fixed relative to said lid.
 19. The system of claim 11, wherein said substrate is configured to be rotatable relative to said chamber.
 20. The system of claim 1, wherein said detection unit comprises one or more optics.
 21. The system of claim 1, wherein said detection unit comprises a sensor configured to capture a signal from said analyte.
 22. The system of claim 1, wherein said chamber is not in mechanical contact with said lid.
 23. The system of claim 1, wherein said lid is configured to move relative to said chamber, or vice versa.
 24. The system of claim 1, wherein said fluid flow unit is configured to maintain said first region at said first atmosphere while said detection unit and said substrate are undergoing motion relative to one another.
 25. The system of claim 1, wherein said fluid flow unit is configured to generate negative pressure in said location disposed between said chamber and said lid.
 26. The system of claim 1, wherein a first portion of said lid is provided between said first region and said second region, and wherein a second portion of said lid is provided between said second region and a third region, wherein a second fluid flow unit is configured to provide fluid in bulk motion to maintain said third region at a third atmosphere that is independent of said first atmosphere and said second atmosphere, and wherein said third region is movable relative to said lid independent of said first region.
 27. The system of claim 1, wherein said second atmosphere is a room atmosphere or an ambient atmosphere.
 28. The system of claim 1, further comprising a controller operatively coupled to said fluid flow unit, wherein said controller is configured to direct said fluid flow unit to cause said fluid to undergo said bulk motion. 